- 702694 - Invitrogen Antibodies LOXL2 antibody

Cummins KA, Bitterman PB, Tschumperlin DJ, Wood DK
APL bioengineering 2021 Dec;5(4):046102

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of LOXL2 was achieved by transfecting U87MG cells with LOXL2 specific validated siRNA (Silencer® select Product # s8261). Western blot analysis (Fig a) was performed using Modified Whole cell extract (1% SDS) from the LOXL2 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-LOXL2 Recombinant Rabbit Monoclonal Antibody (Product # 702694, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to LOXL2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), MDA-MB-231 (Lane 2) and A549 (Lane 3). The blots were probed with Anti-LOXL2 Recombinant Rabbit Monoclonal Antibody (Product # 702694, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 87 kDa bands corresponding to LOXL2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of endogenous LOXL2 using Anti-LOXL2 Recombinant Rabbit Monoclonal Antibody (Product # 702694, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of LOXL2 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating perinuclear localization of LOXL2. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIG. 2. Amplified ECM remodeling occurs after fibroblast activation in collagen microtissues. (a) Lysl oxidase like protein 1 and (b) protein 2 (yellow) were stained and quantified. Mean fluorescence values were normalized by cell number [quantified by nuclear count (pink)]. (c) After 4 days of culture, fibroblasts were stained for procollagen 1alpha2 (yellow), a proxy indicating collagen I synthesis, and Hoechst to visualize nuclei and obtain a cell count for normalization. (d) NHLFs encapsulated in microtissues secreted MMP-2, quantified with an ELISA, and normalized by cell number. Experiment performed three times, with the average of each replicate depicted with a circle; SEM (scanning electron microscopy) shown (pink) (scale 100 mu m; points represent individual microtissues from single experiment).

702694